A deadly protein, perforin, is stored in the granules of cytotoxic lymphocytes and is released to kill virally infected cells and tumor cells. Two proteins, calreticulin and Chymase 1, have opposing influences on the critical pore-forming activity of perforin. Recently, the Hudig lab reported that calreticulin (which is also found in the endoplasmic reticulum) inactivates perforin lysis. Calreticulin binds to membranes and reduces their susceptibility to perform lysis. The lab also observed that Chymase 1 cleaves calreticulin, suggesting that Chymase 1 directly counteracts the effect of calreticulin. Chymase l is the only one of ten granzymes to cleave intact calreticulin. Based on their discoveries, they propose an "inactivation of the inactivator" hypothesis in which calreticulin inactivates perforin lysis and is itself inactivated by Chymase 1 (or its human ortholog). In the scenario, perforin can function independently of calreticulin but, when calreticulin is present, a 'calreticulinase' is needed for lysis. In the cells, lysis will be regulated by the balance of the enzyme Chymase 1 and its substrate calreticulin. The specific aims of the project are: 1) To assess proteolysis of calreticulin after exocytosis from killer cells and how proteolysis affects its ability to regulate perform pore formation; 2) To predict how long intact calreticulin would persist and inactive perforin after exocytosis; 3) To characterize how the binding of calreticulin to membranes changes after its cleavage, including binding to membranes of endosomes that transport Gr B to cause apoptosis; 4) To assess the capacity for intact calreticulin to inactivate cellular cytotoxicity by introducing nonhydrolyzable calreticulin genetic variants (with Ala substitutions at a Tyr or Phe) and by verifying that, when Chymase 1 (and lysis) are inhibited, calreticulin remains intact; and 5) To evaluate mechanisms by which the intact substrate, calreticulin, inactivates perforin lysis. The research will define how a new enzyme (Chymase 1) and its substrate (calreticulin) alter lymphocyte cytotoxic activity. Either enzyme or substrate could be manipulated for therapeutic purpose.